9-purinyl phosphonic acid derivatives

ABSTRACT

This invention relates to novel purine nucleoside phosphorylase inhibitors, to the methods and intermediates for their preparation and to their use as immuno-suppressants, antilymphoma, antileukemic, antiviral and antiprotozoal agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a division of application Ser. No. 08/336,703, filed Nov. 8,1994, which is a continuation of application Ser. No. 07/721,791 filedJun. 26, 1991, now abandoned which is herein incorporated by reference.

FIELD OF THE INVENTION

This invention relates to novel purine nucleoside phosphorylasinhibitors, to the methods and intermediates for their preparation andto their use as immuno-suppressants, antilymphoma, antileukemic,antiviral and antiprotozoal agents.

BACKGROUND

Purine nucleoside phosphorylase (PNP) under normal in vivo conditionscatalyzes the phosphorolytic cleavage of the ribo- anddeoxyribonucleosides of guanine and hypoxanthine to the correspondingsugar phosphate and guanine or hypoxanthine. In the absence of PNP, uricacid concentration is quite low while the concentration of certainnucleoside substrates of PNP such as (dGuo) in plasma and urine areelevated. dGuo is toxic towards lymphoblasts, however, T-cells are muchmore affected than are B-cells. Indeed, in patients with geneticallyacquired PNP deficiency, B-cell immunoglobulin production is normal oreven elevated, but these patients are leukopenic and T-lymphocyticfunction is either totally lacking or is severely depressed. Whileuncontrolled PNP deficiency is obviously undesirable, there are manyinstances where controlled suppression of the immune system, and inparticular controlled supression of T-cells, would be highly desirablesuch as in the treatment of T-cell leukemia, the suppresion ofhost-vs-graft response in organ transplant recipients, and the treatmentof gout. Applicants have discovered a class of 9-purinyl phosphonic acidderivatives which are potent inhibitors of PNP and are thus useful asimmunosuppressant agents.

SUMMARY OF THE INVENTION

More specifically this invention relates to novel purinyl phosphonicacid derivatives of the formula ##STR1## the tautomeric forms thereof,and the pharmaceutically acceptable salts thereof, wherein ##STR2## withAr being a bridging moiety to which its adjacent CH₂ moiety is bonded toone ring carbon atom and the Z moiety is bonded to a second ring carbonatom of an R₉ -substituted phenyl, thiophene or furan moiety, Z is amoiety of sub-types (a), (b), (c), (d), or (e) wherein

(a) is ##STR3## (b) is ##STR4## (c) is ##STR5## (d) is --C.tbd.C--, ₋₋nd (e) is ##STR6## with the proviso that when Z is a sub-type (b)moiety, then Ar is other than a furan or thiophene moiety, n is aninteger of 1 to 5 or zero,

R₁ is --OH or --SH,

R₂ is H or --NH₂,

R₃ is H, --NH₂, --OH or --NH--NH₂,

R₄ is H,

R'₄ is H OH or F or R₄ and R'₄, together with the carbon atom to whichthey are attached, form a keto moiety,

R₅ is C₁₋₆ alkyl or R'₅,

R₆ is C₁₋₆ alkyl or R'₆ with R'₅ and R'₆ being H each of

R₇ and R₈ is H, F, or C₁₋₄ alkyl,

R₉ is H, Cl, Br, C₁₋₆ alkyl, C₁₋₆ alkoxy, OH, NH₂ or CH₃ with theproviso that when Ar is furan or thiophene, R₉ is other than OH or NH₂,

X and Y are H, F or Cl, with the proviso that when n is zero X and Y areboth H.

As used herein the terms C₁₋₄ or C₁₋₆ alkyl include the straight andbranched saturated lower aliphatic hydrocarbyl moieties having up to 4or 6, respectively, carbon atoms including methyl, ethyl, propyl,isopropyl, sec-butyl, n-butyl, t-butyl, pentyl and the like; the C₁₋₆alkoxy moieties being ether derivatives thereof. The "Ar" moietybridging its contiguous CH₂ and Z moieties are R₉ -substituted phenyl,furan or thiophene moieties wherein phenyl may be bridged at its 1,2-,1,3- or 1,4-positions, and each of furan and thiophene may be bridgedthrough the 2,3-, 2,4-, 2,5, or 3,4-position ring carbon atoms; the R₉-substitution may be mono- or di-substituted, substitution being at anyof the other available ring carbon atoms. Tautomeric enol-keto forms mayexist at the 6-position of the purine nucleus.

The expression "pharmaceutically acceptable acid addition salts" isintended to apply to any non-toxic organic or inorganic acid additionsalts of the base compounds of formula 1. Illustrative inorganic acidswhich form suitable salts include hydrochloric, hydrobromic, sulfuric,and phosphoric acids and acid metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids which form suitable salts include the mono-, di-, andtricarboxylic acids. Illustrative of such acids are, for example,acetic, glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric,malic, tartaric, citric, ascorbic, maleic, hydroxymaleic, benzoic,hydroxybenzoic, phenylacetic, cinnamic, salicylic, and 2-phenoxybenzoicacids. Other organic acids which form suitable salts are the sulfonicacids such as methane sulfonic acid and 2-hydroxyethane sulfonic acid.Either the mono- or the di-acid salts can be formed, and such salts canexist in either a hydrated or a substantially anhydrous form. The acidsalts are prepared by standard techniques such as by dissolving the freebase in aqueous or aqueous-alcohol solution or other suitable solventcontaining the appropriate acid and isolating by evaporating thesolution, or by reacting the free base in an organic solvent in whichcase the salt separates directly or can be obtained by concentration ofthe solution. In general the acid addition salts of the compounds ofthis invention are crystalline materials which are soluble in water andvarious hydrophilic organic solvents and which in comparison to theirfree base forms, demonstrate higher melting points and an increasedstability.

The preparation of the compounds of formula I may, in general, beeffected by a condensation reaction wherein a 6-chloro purine (2) istreated with an activated (--CH₂ --Ar--Z--)-substituted phosphonate (3)and the resulting Ia intermediate converted to the appropriate R, R₁,R₂, R₃ -substituted purine derivatives of formula I. The generalcondensation reaction is depicted in the following reaction scheme.##STR7## wherein R₁, R₂, R₃, Ar, Z, R₅ and R₆ are as previously defined,except that R'₄ which occurs when Z is a moiety of sub-type (a) may alsobe a silyl ether, and Q is bromo, iodo or hydroxy.

In those instances wherein it is desired to effect a condensation of the6-chloro purine intermediate (2) with a phosphonoaryl (3) wherein Q is ahalide, the condensation is effected by reacting a slight excess (about10%) of the 6-Cl purine reactant (2) in the presence of a base such assodium hydride (NaH), potassium carbonate (K₂ CO₃) or cesium fluoride(CsF) (in amounts of about 2 equivalents) in a non-reactive solvent suchas dimethylformamide (DMF) within a temperature range of about 0° to 60°C., preferably at room temperature for about 4 to 18 hours.

In the instance wherein Q is OH, the condensation is effected under moreneutral conditions according to the Mitsunobu-type reaction usingdiethyl azodicarboxylate (DEAD) in the presence of P(R')₃ wherein R' ispreferably phenyl but including methyl and isopropyl, said reactionbeing conducted in a suitable non-reactive solvent at 0° to 60° C.

Of course in each instance, in those reactions wherein the Ar moietybears an R₉ -substitutent which may be affected by the reactionconditions of these condensations (or modifications of the purine base)then such substituents are modified to obviate any undesiredside-reactions and, at the appropriate step, are reconverted back to thedesired form. For example, if R₉ is OH, then an intermediate ester orether derivative can be formed, and at the appropriate step, the esteror ether may be hydrolyzed back to its alcohol. These principles arewell-understood by those of ordinary skill in the art and need not bedetailed herein.

In the special instance wherein R₄, R'₄, X and Y of formula (3) are allhydrogen, it is preferred to condense a bromomethyl derivative of thephosphonylaryl of the formula ##STR8## with the purine (2) andhydrogenate the resulting products preferably using hydrogen gas in thepresence of palladium on carbon (H₂ -Pd/C) according to standardtechniques.

In the special instance wherein it is desired to prepare compounds offormula Ia wherein R₄ is H and R'₄ is OH or R₄ and R'₄ form a ketomoiety (as defined above) it is prepared to use a silyl ether,(preferably t-butyl dimethylsilyl ether), to protect the hydroxy moietyof the phosphonyl halide, i.e., ##STR9## wherein ##STR10## represents at-butyl dimethylsilyl ether and following the condensation, aspreviously described, remove the silyl protecting group by acidhydrolysis. If the resulting alcohol is to be oxidized, the alcohol canbe oxidized to the desired ketone by use of the Swern oxidationreaction. In practice it is preferred to form the silyl ether prior toactivating the reactants of formula 3, as herein below described.

The "Q-activated" reactants of formula 3 may be prepared by methods wellknown in the art, preferably utilizing intermediates wherein the OHgroups (if any) are reaction-protected prior to activation with eitherthe halo or hydroxy moieties.

Preferably, brominations are effected with N-bromosuccinimide (NBS) orother suitable N-bromo amides in the presence of catalytic quantititesof benzoyl peroxide, the reaction preferably run in CCl₄ (carbontetrachloride) as solvent. Preparation of the reactants of formula 3wherein Q is OH may be effected directly from (6) by reaction withCeAmNO₃ (ceric ammonium nitrate) or by converting the benzyl bromide (7)to its acetate followed by hydrolysis of the acetate with catalyticamounts of sodium methoxide in methanol, said reaction using standardprocedures well known in the art. ##STR11## wherein Ar, Z, R₅ and R₆ areas previously defined except that R'₄ is a silyl ether (instead of OH)and R₉ is a protected hydroxy group rather than OH (if appropriate).

Once condensation of the 6-chloro purine base (2) is effected to producecompounds of formula Ia, the modifications at the 8,6- and/or2-positions may be effected in a stepwise fashion to produce the desiredR₁, R₂ and R₃ moieties of formula I.

To prepare compounds of formula I wherein both R₅ and R₆ are H and R₁ isOH, the corresponding phosphonate di-esters of Ia (i.e. R₅ and R₆ arealkyl) are successively reacted with trimethylsilylbromide (TMSBr) inCH₂ Cl₂, water in acetonitrile and finally in HCl (IN) at 90° C. Toprepare a monoester (R₅ is H and R₆ is alkyl) and R₁ is OH, thecompounds of Ia are submitted directly to HCl/H₂ O hydrolysis at 90° C.

To prepare compounds of formula I wherein R₁ is SH, compounds of formulaIa are reacted with thiourea in acetic acid. De-etherification of theresulting 6-SH products by treatment with TMSBr and hydrolysis willyield compounds wherein R₁ is SH and R₅ and R₆ are H.

To prepare the compounds of formula I wherein R₁ is SH, R₂ and R₃ are asdefined (formula I) and R'₄ is H or a silyl ether (--O--SiMe₃), thecorresponding 6-OH analog is reacted with dimeric phosphorouspentasulfide. The resulting compounds wherein R'₄ is a silyl ether canbe converted to its alcohol and, if desired, the alcohol can be oxidizedto its keto analog by methods described herein. The same reactants(except that R₃ is H) can be utilized to prepare compounds of formula Iwherein R₃ is NH₂ or --NH--NH₂ by halogenation (at the 8-position of thepurine moiety) using a brominating or iodinating agent such as brominein water, a N-bromo or N-iodo imide (e.g.1,3-dibromo-5,5-dimethyl-hydantoin, 1,3-diiodo-5,5-dimethyl-hydantoin,N-iodoacetamide, preferably NBS or NIS, and most preferablyN-bromoacetamide (NBA). The so produced 8-halo analogs is reacted withhydrazine in a suitable solvent (e.g., water, ether, THF, p-dioxan,lower alkanols, ethylene glycol, chlorinated hydrocarbons (CCl₄, CH₂Cl₂), DMF, HMPA or DMSO at temperatures of about 50°-100° C., preferablybut not necessarily using 2 to 3 fold excess of hydrazine. Thecorresponding 8-NH₂ compounds may be prepared by reducing the hydrazineusing Raney Nickel. The 8-halo analogs used for reaction with hydrazinemay be utilized to prepare compounds wherein R₃ is OH by reacting the8-halo compound with an alkali metal or alkaline earth metal salt of abenzyl alcohol (e.g., benzyl alcohol) followed by subsequent reductionof the intermediate with hydrogen gas at atmospheric pressure in thepresence of a noble metal catalyst (e.g., Pd/C).

To visualize the concepts relating to the route of synthesis and to morereadily teach the alternate pathways by which the compounds of sub-type(a) wherein R₄ is H and R'₄ is OH, or R₄ and R'₄ form the defined ketomoiety, the following schematic is depicted. ##STR12## wherein ##STR13##represents t-butyldimethylsilyl and Ar, X, Y, Bu₄ NF, TMSBr being aspreviously defined. The preparation of the arylphosphonates of formula 6may be effected using standard processes and techniques analogouslyknown in the art; the particular route of synthesis, of course, beingdependent on the definition of the Z moiety.

In the instance wherein it is desired to prepare intermediates offormula 6 wherein Z is represented by sub-type (a), i.e., compounds offormula ##STR14## wherein Ar, R₄, R'₄, X, Y, R'₅ and R₆ are as describedin formula I, the specific route of synthesis is primarily dependent onthe specific definitions of R₄, R'₄, X and Y. In the instance wherein R₄and R'₄ are hydrogen, the procedure is schematically represented asfollows: ##STR15## with M being Li, Na, --ZnBr, MgBr (preferablylithium) and X and Y are H, F or Cl. In this reaction the lithioderivative (17) prepared by reaction of the appropriate phosphonate withlithium di-isopropylamine (LDA) or butyl lithium, under an inertatmosphere (argon) in an anhydrous solvent (e.g. THF) at about -78° C.is condensed with the aryl bromide (16) by reaction for 10-20 hours andthe reaction quenched with saturated aqueous ammonium chloride (NH₄ Cl).Condensation of the reactants (16 and 17) wherein M is --ZnBr ispreferably effected in the presence of catalytic amounts of copperbromide at 20° C. The Zn and Mg bromide derivatives are also prepared bystandard processes.

In the instance wherein R₄ is H and R'₄ is OH or F, or R₄ and R'₄ form aketo moiety (as defined), the compounds are prepared as schematicallyillustrated as follows: ##STR16##

The reaction of the aldehydes (18) with the lithio derivative (19) iseffected in THF at -78° C. under argon for about 3 hours and thereaction is then quenched with saturated aqueous NH₄ Cl at about -78° C.to -30° C. to produce compounds (20). In treating compounds (20) withDAST (diethylamino sulfurtrifluoride) the reaction is run indichloromethane at about 0° C. for about 15 to 25 hours and the reactionquenched using excess methanol. The alcohols (20) may also be oxidizedto their keto forms by use of the Swern oxidation reaction using oxalylchloride in DMSO) or by the use of tetrapropylammonium perruthenate andN-methyl morpholine N-oxide. Alternatively, the ketones may be formeddirectly from reaction of the compounds (19) (wherein M is Li or --ZnBr)with compounds of the formula ##STR17## wherein X" is chloro or alkoxy.

In effecting this latter reaction to prepare the ketones directly, it isspecifically preferred that X" be alkoxy (e.g., methoxy) and M belithium when both X and Y are H, and when both X and Y are F it isspecifically preferred that X" be chloro and M be --ZnBr.

In the case wherein R₄ is H, R'₄ is OH or R₄ and R'₄ form the definedketone, it is preferred that a silyl ether of compound (20) be utilized(e.g., a t-butyl dimethylsilyloxy derivative) as already described forthe condensation of the reactants (2) and (3). These silyl derivativesare prepared by reacting compounds 19 with t-butyl dimethylsilylchloride in the presence of imidazole and the resulting productsactivated with NBS. Selective removal of the silylprotecting group at alater stage is effected by treatment with tetrabutyl ammonium fluoride(Bu₄ NF), as particularized above.

In the particular case when compounds of Ia are prepared wherein R₄ andR'₄ form the defined ketone and X and Y are H, it is preferred toutilize the following reaction scheme: ##STR18##

It should be noted that in the instance wherein it is desired to preparecompounds of Ia wherein Z is sub-type (a) by a condensation wherein Q offormula 3 is OH, and each of R₄, R'₄, X, and Y are H, it is preferred toreduce intermediates of sub-type (c) to obtain the appropriate reactantsembraced within generic formula 3.

In the instance wherein it is desired to prepare intermediates embracedwithin formula 6 wherein Z is of sub-type (b), i.e., compounds offormula ##STR19## wherein Ar' is other than a furan or thiophenebridging moiety, standard procedures analogously known in the art may beutilized. In general, the intermediates are prepared according to thefollowing reaction scheme: ##STR20## wherein R'₉ is as defined for R₉ informula I other than OH, Q' is iodo, bromo, tosylate, mesylate ortriflate leaving groups and n is an integer 1 to 5. The condensation iseffected in the presence of a base (e.g., NaH, K₂ CO₃ or KH) in anon-aqueous solvent (e.g., DMF, THF or DMSO) using standard procedureswell known in the art. In the special instance wherein n is 2, thecresols (i.e., o, m, or p cresols) are reacted with ethylene carbonatein the presence of KF to obtain the benzoxy ethyl-1-ol-ether which isconverted to its 1-bromo derivative by reaction with bromine in thepresence of triphenyl phosphine (Po₃) in benzene in the presence of abase to afford the benzoxy ethyl bromide, by standard procedures isreacted with a lithio derivative of formula (17) to produce compounds(30a) wherein n is 2.

Of course, when n is zero and X and Y are both H, it is preferred to usethe process wherein Q' is a tosylate (of compounds 29) using NaH as abase in DMF.

In those instances wherein it is desired to prepare compounds of formula6 wherein Z is represented by sub-type (c), i.e., compounds of theformula ##STR21## wherein Ar, X, Y, R₅ and R₆ are as defined as informula I, said compounds may be prepared by methods and processesanalogously known in the art.

In the instance wherein Y is H and X is H, F or Cl, an aryl aldehyde(32) is condensed with a lithio derivative of an X-substituteddiphosphonate derivative (33) according to the reaction scheme:##STR22##

The reaction is conducted at -78° C. in THF and prior to quenching themixture is allowed to warm to about 20° C. before hydrolysis withsaturated aqueous NH₄ Cl. When Y is F and X is H, F, or Cl, thepreparation is effected using a compound of sub-type (a) (i.e., compound(35) wherein X' is F or Cl) which is treated with a base, preferablytBuOK, DBU of DMAP in a non-reactive solvent (DMF or DMSO) attemperatures of about 40° C. to 80° C. In this reaction the double bondis created by the loss of HX'. By choosing the appropriate analogs ofcompounds (35) and by following the foregoing treatment with a base, thedesired Y and X compounds (34a) may be prepared when either HF or HCl issplit off. The reaction is depicted as follows: ##STR23##

In the instance wherein it is desired to prepare intermediates offormula 6 wherein Z is represented by sub-typed (d), i.e., compounds offormula ##STR24## the compounds are prepared by treatment of compoundsof formula (24) with two equivalents of DAST at 0° C. to 20° C. in CH₂Cl₂ for 1 to 5 hours and the reaction is quenched with excess methanolto yield the desired products 36.

In those instances wherein it is desired to prepare compounds of formula6 wherein Z is represented by sub-type (e), i.e., compounds of theformula ##STR25##

Their preparation may be effected in a one or two step olefinationprocess using compounds of formula 38 ##STR26##

In the one-step process a Wittig-type olefination process using aphosphonium ylide of the formula ##STR27## reacted with a compound offormula 38 (especially when X and Y are F); the reaction being conductedin THF at -78° C. to 0° C. yield the double bond. Alternatively reactant39 may be replaced with a reactant (40) of the formula ##STR28## whereinHet is --So, SiMe3, Seo, --SMe or SeMe, and when reacted will producecompounds of formula (41) ##STR29##

In those instances wherein Het is SiMe₃, the Peterson olefination isutilized using (a) treatment of 41 with NaH in DMF at 0° to 60° C. or(b) treatment of 41 with an acid, e.g., PTSA (p-toluenesulfonic acid) atelevated temperatures. When het is other than SiMe₃, treat 41 withSOCl₂, POCl₃ or PI₃ with Et₃ N or pyridine in CH₂ Cl₂ at temperature ofabout -20° to 20° C.

The following examples will illustrate the methods by which thecompounds of this invention may be prepared.

SYNTHESIS OF 1[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoroethyl]phosphonicacid PREPARATION OF 1A: [2-[2-methylphenyl]-1,1-difluoroethyl]phosphonicacid, diethyl ester difluoroethyl]phosphonic acid, diethyl ester

30 mmol (5.64 g) of diethyl difluoromethane phosphonate dissolved in 30ml of anhydrous tetrahydrofuran (THF) are slowly added to a stirredsolution of (LDA) lithium diisopropylamide (prepared at 0° C. from 31mmol of n-butyllithium and 30 mmol of diisopropylamine in 30 ml ofanhydrous THF) at -78° C. under argon. After 30 minutes, 45 mmol (8.33g) of 2-bromo-o-xylene are added to the reaction mixture which isstirred at -78° C. for 15 hours and quenched by adding 20 ml of anaqueous saturated solution of ammonium chloride. The crude mixture isevaporated to dryness; the residue is suspended in 50 ml of water andextracted 3 times with 100 ml of ethylacetate. The organic layers aredried over sodium sulfate, filtered and evaporated to give approximately8 g of crude product which is purified by flash chromotography on silicagel affording 3.6 g of 1A (41% yield).

PREPARATION OF 1B:[2-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenyl]-1,1,difluroroethyl]phosphonicacid, diethyl ester

N-bromo-succinimide (3 mmol, 0.53 gr) and benzoyl peroxide (5 mg) areadded to a solution of 1A (3 mmol, 0.88 g in 20 ml of carbontetrachloride). The mixture is refluxed, by heating with a lamp for 90minutes, until all solid succinimide is apparent. The reaction mixtureis filtered to remove succinimide and the filtrate is evaporated todryness to give 1.1 g of an oil which is then added to a stirredsolution of the sodium salt of 6-chloroguanine (prepared in 5 ml DMF byadding 3,2 mmol of sodium hydride to 3.9 mmol of 6-chloroguanine at 20°C. under argon). The reaction mixture is stirred at 20° C. for 20 hours,evaporated under reduced pressure and purified by flash chromotographyon silica gel giving 1.15 g of expected 1B (42%).

PREPARATION OF 1:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoroethyl]phosphonic acid

4 mmol (0.5 ml) of trimethylsilylbromide (TMSBr) are added to a stirredsolution of 1C (0.6 g; 1.3 mmol) in 5 ml of anhydrous dichloromethane at20° C. under argon. The reaction mixture is stirred for 20 hours and 0.5ml of TMSBr is added to the reaction mixture. After 20 hours, thereaction mixture is evaporated; the residue is dissolved in 3 ml ofacetonitrile and quenched with approximately 0.2 ml of water.

The mixture is evaporated and the residue is heated in 7 ml of 1N HCl at100° C. for 20 hours. The mixture is evaporated and the product isobtained after 2 recrystallizations from hot water: 200 mg (38% yield;mother liquors contain essentially pure product for subsequentisolation.)

SYNTHESIS OF 2[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-methyl]phenyl]-1-fluoroethenyl]phosphonicacid PREPARATION OF 2A: [2-(2-methylphenyl]-1-fluoroethenyl]phosphonicacid, diethyl ester

20 mmol (6.72 g) of bis(diethylphosphonyl)fluoromethane dissolved in 20ml of anhydrous THF are slowly added to a -78° C. solution of lithiumdiisopropylamide (prepared by adding 22 mmol of n-butyllithium to asolution of 22 mmol of diisopropylamine in 16 ml of THF at 0° C.). After30 minutes at -78° C., a solution of 30 mmol (3.5 ml) of freshlydistilled o-tolualdehyde in 20 ml of THF is added to the reactionmixture which is stirred at -78° C. for 3 hours and at 20° C. for 5hours, quenched with 20 ml of aqueous saturated ammonium chloride andevaporated to dryness. The residue is suspended in 30 ml of water andextracted three times with 100 ml of ethylacetate. The organic layersare washed with brine, dried over sodium sulfate, filtered, andevaporated to give 5 g of crude material which is purified by flashchromatography on silica gel affording 70 mmol (50%) of 2A.

PREPARATION OF 2D:[2-[2-(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenyl]-1-fluoroethyenyl]phosphonicacid, diethyl ester

Benzoyl peroxide (10 mg) is added to a suspension of NBS (10 mmol) and2A (10 mmol) in 15 ml of anhydrous carbon tetrachloride. The mixture isheated to reflux with a lamp until all solid is floating up. Thereaction mixture is filtered and evaporated to give 2C as an oil whichis then dissolved in 4 ml of anhydrous DMF and added to a stirredsolution of the sodium salt of 6-chloro-guanine (prepared by adding 10mmol of NaH (as a 60% solution w/v) to 10 mmol of 6-chloro-guanine in 10ml of anhydrous DMF at 20° C. under argon). After 20 hours at 20° C.,the reaction mixture is evaporated to dryness and the crude residue isdirectly purified by flash chromatography on silica gel giving 4 mmol ofproduct 2D (40% yield).

PREPARATION OF 2:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1-fluoroethenyl]phosphonic acid

The preparation of 2 from 2D was performed using the procedure alreadydescribed for the transformation of 1C into 1.

SYNTHESIS OF 3

[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1,2-trifluoroethyl]phosphonicacid PREPARATION OF 3A:[2-hydroxy-2-(2-methylphenyl]1-difluoro-ethyl]phosphonic acid, diethylester

42.5 mmol of diethylphosphinyl difluoromethane (8 g) dissolved in 42 mlof anhydrous THF are slowly added to a stirred solution of freshlyprepared lithium diisopropylamide (42.5 mmol) in 40 ml of THF at -78° C.under argon. The reaction mixture is stirred at -78° C. for 35 minutesand 7.65 g of o-tolualdehyde (63.75 mmol) dissolved in 42 ml of THF areadded to the reaction mixture which is stirred at -78° C. for 4 hours,quenched at -78° C. by addition of 40 ml of saturated aqueous ammoniumchloride and evaporated under reduced pressure. The residue is suspendedin water and extracted three times with 200 ml portions of ethylacetate. The organic layers are washed with brine, dried over sodiumsulfate, filtered, evaporated and purified by flash chromatography onsilica gel to give 10.67 g of 3A as white crystals (81% yield)

PREPARATION OF 3B: [2-fluoro-2-[2-methylphenyl]-1,1-difluoroethyl]phosphonic acid, diethylester

2.3 ml of diethylamino sulfur trifluoride (DAST) are added dropwise to astirred solution of 3A (4.6 g; 15 mmol) in 20 ml of anhydrousdichloromethane at 20° C. under argon. After two hours at 20° C., thereaction mixture is slowly quenched at 0° C. with excess methanol (5ml), evaporated to dryness and directly purified by flash chromatographyon silica gel to give 4.22 g of product 3B (91% yield).

PREPARATION OF 3C:[2-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)Methyl]phenyl]-1,1,2-trifluoroethyl]phosphonicacid, diethyl ester

The bromination reaction of 3B and subsequent condensation with6-chloroguanine was run exactly as described for the preparation of 1Cfrom 1B.

PREPARATION OF 3:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1,2-trifluoroethyl]phosphonicacid

Final product 3 was isolated after deprotection with TMSBr/CH₂ Cl₂ and1N HCl in water as described for the preparation of 1 from 1C.

SYNTHESIS OF 4[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)-methyl]phenyl]-2-hydroxy-1,1-difluoroethyl]phosphonicacid PREPARATION OF 4A:[2-(t-butyldimethylsilyloxy)-2-(2-methyl-phenyl)-1,1-difluoroethyl]phosphonicacid, diethyl ester

Diethylphosphinyl difluoromethane (4.7 g, 25 mmol) dissolved in 25 ml ofanhydrous THF is added dropwise to a -78° C. solution of lithiumdiisopropylamide (LDA) (prepared by reacting 25 mmol of n-butyllithiumwith 25 mmol of diisopropylamine at 0° C. in 25 ml of THF) under argon.After minutes at -78° C., a solution of o-tolualdehyde (30 mmol, 3.6 g)in 20 ml of anhydrous THF is added to the reaction mixture. After 3hours at -78° C., 30 mmol of t-butyldimethylsilylchloride are added tothe reaction mixture which is stirred at -20° C. for 2 hours, quenchedwith 10 ml of water, evaporated, extracted 3 times with 120 ml portionsof ethylacetate. The organic layers are gathered, dried over sodiumsulfate, filtered, evaporated and purified by flash chromatography onsilica gel to give 8.8 g of product 4A (21 mmol, 84% yield).

PREPARATION OF 4B:[2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-[2-(t-butyldimethylsilyloxy)-1,1-difluoroethyl]phosphonicacid, diethyl ester

The preparation of 4B from 4A was performed using the procedure alreadydescribed for the transformation of 1A into 1C.

PREPARATION OF 4C:[2-[2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2-hydroxy-1,1-difluoroethyl]phosphonicacid, diethyl ester

12 mmol (3.85 g) of tetrabutyl ammonium fluoride are added in oneportion to a stirred solution of 4B (6 mmol) dissolved in 150 ml of THF.The reaction mixture is stirred at 20° C. for 20 hours, evaporated todryness and purified by flash chromatography on silica gel giving 4.8mmol of 4C (80% yield).

PREPARATION OF 4:[2-[2-[(2-amino-1,6-dihydro-6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2-hydroxy-1,1-difluoroethyl]-phosphonicacid

Compound 4 was obtained from 4C by the two chemical deprotection steps(TMSBr); H₃ O⁺) already described.

SYNTHESIS OF 5[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]-phenyl]-2,2-dihydroxy-1,1-difluoroethyl]phosphonicacid PREPARATION OF 5A FROM 4C:[2-[2-(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2-hydroxy-1,1,difluoroethylphosphonic acid, diethyl ester

60 mmol (4.3 ml) of DMSO dissolved is 25 ml of anhydrous dichloromethaneare added dropwise at -65° C. to a stirred solution of 30 mmol ofoxalylchloride (26 ml) in 25 ml of anhydrous dichloromethane underargon. The reaction mixture is stirred at -65° C. for 5 minutes and 20mmol of 4C dissolved in 25 ml of CH₂ Cl₂ are added. The reaction flaskis removed from the cooling bath for a few minutes and the mixture isstirred again at -65° C. for 15 minutes. At that time, 100 mmol (13.8ml) of triethylamine are added to the reaction mixture which is stirredfor 10 minutes at -65° C, quenched with aqueous citric acid, stirred afew minutes at 20° C., extracted with dichloromethane (3 times with 75ml portions), washed with brine, dried over Na₂ SO₄, filtered,evaporated, and purified by flash chromatography on silica gel giving13.5 mmol of product 4C (67%).

PREPARATION OF 5:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2,2-dihydroxy-1,1-difluoroethyl]phosphonicacid

Final product 5 is obtained from 5A by two deprotection steps performedas in the preparation of 1 from 1C.

SYNTHESIS OF 6[3-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenoxy]-1,1-difluoropropyl]phosphonicacid PREPARATION OF 6A: 3-(2-methylphenoxy)-1-propanol

300 mmol of ortho-cresol, 334 mmol of ethylene carbonate and 325 mmol ofpotassium fluoride are added to 100 ml of anhydrous DMF and stirred at125° C. for 50 hours under argon. 40 mmol of ethylene carbonate and 40mmol of KF are added to the reaction mixture which is then stirred foranother 24 hours at 125° C.

The reaction mixture is cooled to 20° C., filtered, and evaporated. Theresidue is purified by flash chromatography on silica gel to give 38.6 gof expected product (85% yield).

PREPARATION OF 6B: 3-(2-methylphenoxy)-1-bromopropane

10 g of bromine (62.5 mmol) dissolved in 30 ml of benzene (oracetonitrile) are slowly added to a stirred solution oftriphenylphosphine (64 mmol) in 100 ml of benzene (or acetonitrile).After 15 minutes, triethylamine (64 mmol) dissolved in 35 ml of benzene(or acetonitrile) is added to the reaction mixture followed by additionof the starting material 6A (9.68 g; 63.7 mmol) dissolved in 50 ml ofbenzene (or acetonitrile). The reaction mixture is stirred at 20° C. for20 hours, filtered (to removed most of the triphenylphosphine oxide),evaporated, and purified by flash chromatography on silica gel to give9.8 g of expected product.

PREPARATION OF 6C: [3-(2-methylphenoxy)-1,1-difluropropyl]phosphonicacid, diethyl ester

30 mmol (5.64 g) of difluoromethyl O,O-diethylphosphonate dissolved in30 ml of anhydrous THF are slowly added to a stirred solution of 37 mmolof LDA (prepared from 31 mmol of n-butyllithium and 31 mmol ofdiisopropylamine in 30 ml of THF) at -78° C. under argon. The reactionmixture is stirred at -78° C. for 30 min and the starting material 6B(20 mmol) dissolved in 10 ml of anhydrous THF is added to the reactionmixture. Stirring is continued for 3 hours at -78° C.; the temperatureis slowly raised to 20° C., and the reaction mixture is quenched withsaturated aqueous ammonium chloride. The crude mixture is thenevaporated and extracted with ethylacetate. The organic layers aregathered, washed with water and brine, dried over sodium sulfate,filtered, evaporated, and purified by flash chromatography on silica gelto give 40% (16 mmol) of the expected condensation product.

PREPARATION OF 6D: [3-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenoxy]-1,1-difluropropyl]phosphonic acid, diethyl ester

6 mmol of starting material 6C dissolved in 15 ml of anhydrous carbontetrachloride are heated with a lamp with 6 mmol of N-bromosuccinimideand a few milligrams of benzoylperoxiole during 35 minutes. The crudemixture is filtered to remove succinimide and the filtrate is evaporatedto dryness, dissolved in 8 ml of anhydrous DMF and stirred at 20° C.under argon with 6.5 mmol of 6-chloroguanine and 13 mmol of potassiumcarbonate for 24 hours. The mixture is evaporated to dryness and theresidue is suspended in 50 ml ethylacetate, washed with ammoniumchloride and brine, dried over sodium sulfate, filtered, evaporated, andpurified by flash chromatography on silica gel to give 3 mmol of theexpected product.

PREPARATION OF 6:[3-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenoxy]-1,1-difluoropropyl]phosphonicacid

9 mmol of freshly distilled TMSBr are slowly added to a stirred solutionof starting material 6D (3 mmol) dissolved in 10 ml of anhydrousdichloromethane at 20° C. under argon. The reaction mixture is stirredat 20° C. for 20 hours and evaporated to dryness. The residue isdissolved in 8 ml of anhydrous acetonitrile and quenched with 10 mmol ofwater. A white precipitate is formed which is separated by filtrationand collected to give the expected product which is used without furtherpurification in the next step.

2 mmol of starting material dissolved in 10 ml of 1N HCl and 2 ml of THFare heated at 90°-100° C. for 20 hours. The reaction mixture is cooledto 20° C., evaporated to dryness, dissolved in saturated aqueoustriethylammonium bicarbonate, filtered and crystallized by addition of1N HCl.

The white solid is collected, dried under reduced pressure to give 1.7mmol of expected product as the hemihydrate.

SYNTHESIS OF 7[2-[(2-amino-1,6,dihydro-6-oxo-9H-purin-9yl)methyl]phenoxy]methylphosphonicacid PREPARATION OF 7A: 2-methylphenoxymethyl phosphonic acid, diethylester

Sodium hydride (8 mmol of a 60% suspension in oil) is added to a stirredsolution of o-cresol (8 mmol, 864 mg) in 10 ml of anhydrous DMF at 20°C. under argon. After 45 minutes, the O,O-diethyl methyl phosphonatetosylate derivative (8 mmol, 2.54 g) dissolved in 3 ml of DMF is addedto the reaction mixture which is stirred at 60° C. for 20 hours,evaporated under reduced pressure and purified by flash chromatographyon silica gel to give 1.1 g of product (69% yield).

PREPARATION OF 7B: [2-[(2-amino-1,6,dihydro-6-chloro-9H-purin-9yl)methyl]phenoxy]methylphosphonic acid,diethyl ester

The phosphonate 7A (1.03 g, 4 mmol), N-bromosuccinimide (4.2 mmol; 743mg) and a few milligrams of benzoyl peroxide in 10 ml of CCl₄ are heatedto reflux under a heating lamp. After 35 minutes, the reaction mixtureis filtered and evaporated to give 1.3 g of an oil which is dissolved in3 ml of anhydrous DMF and added to a stirred suspension of6-chloroguanine (4.4 mmol; 745 mg) and potassium carbonate (10 mmol;1.38 g) in 6 ml of anhydrous DMF at 20° C. under argon. After 40 hours,the reaction mixture is evaporated under reduced pressure and purifiedby flash chromatography on silica gel giving 1.25 g of expected product(74% yield).

PREPARATION OF 7:[2-[(2-amino-1,6,dihydro-6-oxo-9H-purin-9yl)methyl]phenoxy]methylphosphonicacid

By chemical deprotection using TMSBr and then aqueous hydrolysisperformed as already described one obtains the title compound.

SYNTHESIS OF 8[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]1,1-difluoro-2-propenyl]phosphonicacid PREPARATION OF 8A: [2-[2-methylphenyl]1,1difluorooxoethyl]-phosphonic acid, diethyl ester

50 mmol (13,35 g) of O,O-diethylbromodifluoromethane phosphonatedissolved in 50 ml of dimethoxyethane (DME) are slowly added to astirred suspension of freshly activated zinc (55 mmol) in 15 ml of DMEat such a rate that a gentle reflux is maintained. The reaction mixtureis stirred at 20° C. for 2 hours and 60 mmol of o-toluic acid chloride(25 g) dissolved in 15 ml of DME are added to the reaction mixture whichis stirred at 20° C. for 20 hours. The crude material is filtered overcelite and the filtrate is evaporated to dryness and directly purifiedby flash chromatography on silica gel giving 30 mmol of product 8A(60%).

PREPARATION OF 8B: [2-[2-methylphenyl]1,1 difluoro-2-propenyl]phosphonicacid, diethyl ester

35 mmol of n-butyllithium (21.8 ml of 1.6N solution in hexene) areslowly added to a stirred suspension of 35 mmol of methyltriphenylphosphonium bromide in 50 ml of THF at -78° C. under argon. Thereaction mixture is stirred for 2 hours at 0° C., and 30 mmol ofcompound 8A dissolved in 30 ml of THF are added to this reaction mixtureat -78° C.

After stirring at -78° C. for 2 hours and at 0° C. for 2 hours, thereaction mixture is hydrolyzed with saturated aqueous ammonium chloride.The crude product is evaporated under reduced pressure and extracted 3times with 100 ml of ethyl acetate. Usual work-up and purification byflash chromatography on silica gel affords 18 mmol of 8B (60% yield).

PREPARATION OF 8C:[2-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenyl]1,1difluoro-2-propenyl]phosphonic acid, diethyl ester

The bromination reaction of 8B and subsequent condensation with6-chloroguanine was run exactly as described for the preparation of 1Cfrom 1B.

PREPARATION OF 8:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]1,1difluoro-2-propenyl]phosphonic acid

Final product 8 was isolated from 8C after deprotection with TMSBr/CH₂Cl₂ and 1N HCl in water as described for the preparation of 1 from 1C.

SYNTHESIS OF 9[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methylphenyl]ethynyl]phosphonicacid PREPARATION OF 9A: [2-[2-methylphenyl]-2-oxoethane phosphonic acid,diethyl ester

100 mmol of n-butyllithium are slowly added to a stirred solution of 100mmol of methyl phosphonic acid, diethyl ester dissolved in 100 ml of THFat -78° C. under argon. The reaction mixture is stirred at -78° C. for 2hours and 50 mmol of o-toluene acid methyl ester dissolved in 50 ml ofTHF are added to the reaction mixture which is stirred at -78° C. for 20hours and at 0° C. for 2 hours before being hydrolyzed by saturatedaqueous ammonium chloride. Usual work-up and flash chromatography onsilica gel affords 45 mmol (90% yield) of product 9A.

PREPARATION OF 9B: [2-[2-methylphenyl]ethynylphosphonic acid, diethylester

61 mmol (8 ml) of diethylamino sulfurtrifluoride (DAST) are slowly addedto a solution of 30 mmol of 9A in 50 ml of anhydrous dichloromethane at0° C. The reaction mixture is stirred at 20° C. for 30 hours, and slowlyquenched with excess methanol (5 ml) at 0° C. The reaction mixture isevaporated to dryness and directly purified by flash chromatography onsilica gel giving 24 mmol of product 9B (80% yield).

PREPARATION OF 9C:[2-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenyl]ethynyl]phosphonicacid, diethyl ester

The bromination reaction of 9B and subsequent condensation with6-chloro-guanine was run exactly as described for the preparation of 1Cfrom 1B.

PREPARATION OF 9:[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methylphenyl]ethynyl]phosphonicacid

20 mmol (2,5 ml) of TMSBr are added to a stirred solution of 9C (5 mmol)in 25 ml of anhydrous dichloromethane at 20° C. under argon. Thereaction mixture is stirred for 20 hours and evaporated under reducedpressure. The residue is dissolved in 20 ml of acetonitrile and a whitesolid is precipitated by addition of 0.5 ml of water. The white solid iscollected by filtration and dissolved in a mixture of 15 ml of 0.2H HCland 6 ml of THF. This solution is heated at 60° C. for 8 hours and thefinal product 9 is obtained after crystallization on cooling (1.4 mmol,28% yield).

SYNTHESIS OF 102-2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methylphenyl]ethenyl]phosphonicacid PREPARATION OF 10A: [2-[2-methylphenyl]-ethenyl]phosphonic acid,diethyl ester

38 mmol (10.95 g) of bis(diethylphosphonyl)methane dissolved in 25 ml ofanhydrous tetrahydrofuran are slowly added to a suspension of NaH (42mmol) in 20 ml of anhydrous tetrahydrofuran, at -15° C., under argon.After 45 minutes, 38 mmol (4.6 g) of o-tolualdehyde dissolved in 40 mlof tetrahydrofuran are added to the reaction mixture at 0° C. Afterstirring at 20° C. for 18 hours, the crude reaction mixture is quenchedwith 20 ml of aqueous saturated ammonium chloride and evaporated todryness. The residue is suspended in 35 ml of water and extracted threetimes with 100 ml of ethyl acetate. The organic layers are washed withbrine, dried over sodium suflate, filtered and evaporated to give 11 gof crude material which is purified by flash chromatography on silicagel affording 7.53 g of product 10A (75% yield).

PREPARATION OF 10B:[2-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenyl]ethenyl]phosphonicacid, diethyl ester

Benzoyl peroxide (20 mg) is added to a suspension of N-bromosuccinimide(20 mmol) and [2-[2-methylphenyl]-ethenyl] phosphonic acid, diethylester (20 mmol) in 15 ml of anhydrous carbon tetrachloride. The mixtureis heated to reflux with a lamp until all solid is floating up. Thereaction mixture is filtered and evaporated to give an oil which is thendissolved in 10 ml of anhydrous dimethylformamide and added to a stirredsolution of the sodium salt of 6-chloro-guanine (prepared by adding 20mmol of NaH to 20 mmol of 6-chloro-guanine in 10 ml of anhydrousdimethylformamide at 20° C. under argon. The reaction mixture is stirredfor 20 hours at 20° C., evaporated to dryness and the crude residue isdirectly purified by flash chromatography on silica gel giving 12 mmolof product 10B (60% yield).

PREPARATION OF 10:[2-[2[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methylphenyl]ethenyl]phosphonicacid,

The preparation of 10 from 10B is performed using the procedure alreadydescribed for the transformation of 1B to 1 of Synthesis 1.

BIOLOGICAL USEFULNESS

The ability of the compounds of this invention to act asimmunosuppressant, antilymphoma, antileukemic, antiviral, andantiprotozoal agents and as agents useful in the treatment of gout,psoriasis and autoimmune diseases can be demonstrated by their abilityto inhibit purine nucleoside phosphorylase (PNP). Purine nucleosidephosphorylase (PNP) inhibitory activity can be determined by the coupledxanthine oxidase method of Kalckar, using inosine as the substrate [H.M. Kalckar, J. Biol. Chem. 167, 429-443 (1947)]. Apparent dissociationconstants (K_(I)) were measured at 1 mM inorganic phosphate using 0.1MHEPES buffer (pH 7.4), four concentrations of inosine ranging from 0.05mM to 0.15 mM and various concentrations of inhibitor. The K_(i) forrepresentative members of the compounds of formula 1 are tabulated intable 1 and are compared to the K_(M) values of the substrate inosineusing PNP from various sources. Moreover, compounds of this inventionhave been shown to be effective against lymphomas (human MOLT-4 cells)and thus have antilymphomic and antileukemic activities. The presence of2'-deoxyguanosine (about 1-10 μM), a natural metabolite, appears to beimportant for activity against lymphoma cells in culture.

    __________________________________________________________________________                      ##STR30##                                                                            Rat    Human                                         Compound         Bovine Spleen                                                                         Erythrocytes                                                                         Erythrocytes                                                                         E. Coli                                __________________________________________________________________________    [2-[2-[(2-amino-1,6-dihydro-6-oxo-                                                               4 × 10.sup.-9                                                                   2 × 10.sup.-9                                                                 13 × 10.sup.-9                                                                 15 × 10.sup.-9                  9H-purin-9-yl)methyl]phenyl]-                                                 1,1,difluoroethyl]phosphonic acid                                             [2-[2-[(2-amino-1,6-dihydro-6-oxo-                                                               8 × 10.sup.-10                                                                  4 × 10.sup.-10                                                               1.8 × 10.sup.-9                                                                2.5 × 10.sup.-10                 9H-purin-9-yl)methyl]phenyl]-1-                                               fluoroethenyl]phosphonic acid                                                 [2-[2-[(2-amino-1,6-dihydro-6-oxo-                                                               --      --   3.2 × 10.sup.-9                                                                  5 × 10.sup.-10                 9H-purin-9-yl)methyl]phenyl]-                                                 fluoroethenyl]phosphonic acid                                                 [2-[2-[(2-amino-1,6-dihydro-6-oxo-                                                               6 × 10.sup.-10                                                                  5 × 10.sup.-10                                                               1.3 × 10.sup.-9                                                                  7 × 10.sup.-10                 9H-purin-9-yl)methyl]phenyl]-                                                 1,1,2-trifluoroethyl]phosphonic acid                                          [2-[2-[(2-amino-1,6-dihydro-6-oxo-                                                             2.5 × 10.sup.-7                                                                 3.7 × 10.sup.-8                                                                2.1 × 10.sup.-7                                                                  --                                   9H-purin-9yl)methyl]pheno × y]-                                         1,1-difluoropropyl]phosphonic acid                                            [2-[(2-amino-1,6-dihydro-6-oxo-9H-                                                             7.5 × 10.sup.-9                                                                 7.3 × 10.sup.-9                                                                7.9 × 10.sup.-8                                                                4.5 × 10.sup.-9                  purin9yl)methyl]pheno × y]methyl-                                       phosphonic acid                                                               inosine            3 × 10.sup.-5                                                                 1.5 × 10.sup.-4                                                                1.5 × 10.sup.-4                                                                  8 × 10.sup.-5                  __________________________________________________________________________

As used herein the term, patient, in regard to the suppression of immunesystem means mammals such as mice, rats, cats, dogs, cattle, sheep,swine, and primates including humans. The term, patient, in regard tothe treatment of parasitic infections includes not only mammals but alsoother warm blood animals such as fowl including chickens and turkey.

The term protozoa is intended to include those members of the subphylaSarcomastigophora and Sprozoa of the phylum Protozoa. More particularlythe term protozoa as used herein is intended to include those genera ofparasitic protozoa which are important to man because they either causewhich are important to man because they either cause disease in man orin his domestic animals. These genera are for the most part foundclassified in the superclass of Mastigophora of the subphylumSarcomastigophora and the class of Telosporea of the subphylum Sporozoain the classification according to Baker (1969). Illustrative genera ofthese parasitic protozoa include Histomonas, Trypanosoma, Giardia,Trichomonas, Eimeria, Isopora, Toxoplasma, and Plasmodium.

Indeed, a preferred embodiment of the present invention is the use ofthese compounds as antiprotozoal agents in the treatment of intestinalcoccidia in commercial poultry. Intestinal coccidia infections areresponsible for multi-million dollars loses to the poultry industry inthe United States each year. Due to the rapid development of drugresistance by coccidia, and due to the relatively high toxicity of someof the drugs used in the treatment of coccidiosis, there is a need foreffective coccidiostats that are non-toxic and to which intestinalcoccidia do not develop rapid drug resistance.

Although the immune system is a major defense against substances whichcan cause disease, it cannot distinguish between helpful and harmfulforeign substances and destroys both. It would be useful in manyinstances to have a means of regulating the immune system withoutharming the individual. The compounds of this invention exhibit suchmodulating or regulatory effects and have potential for use in thetreatment of various immune disorders such as rheumatoid arthritis andlupus erythromatus.

Circulating antibodies and cellular immune responses play a role in therejection of transplanted tissues and organs. Unless the donor is theidentical twin of the recipient or is the individual himself, therecipient's lymphocytes recognize the transplant as "not self" andimmediately respond to destroy it. The exceptions to this situation aretransplants to non-vascularized areas (privileged sites), such as thecornea of the eye, where lymphocytes do not circulate and therefore arenot sensitized and do not prompt an immune response. It is currentlydifficult to suppress the immune reaction to prevent rejection of thetransplant without severely damaging the patient in other ways. Thepatient must also be given massive doses of antibiotics because his owndefenses against infection have been suppressed. The compounds of thisinvention could be valuable in establishing tolerance to the transplantthrough controlled modulation of the immune system. In addition, thesecompounds demonstrate antiviral activities.

The amount of the active ingredient to be administered can vary widelyaccording to the particular dosage unit employed, the period oftreatment, the age and sex of the patient treated and the nature andextent of the disorder treated. The total amount of the activeingredient to be administered will generally range from about 1 mg/kg to100 mg/kg and preferably from 3 mg/kg to 25 mg/kg. A unit dosage maycontain from 25 to 500 mg of active ingredient, and can be taken one ormore times per day. The active compound of formula 1 can be administeredwith a pharmaceutical carrier using conventional dosage unit formseither orally, parenterally, or topically. In a preferred mode,2-deoxyguanosine will be administered conjunctively with a compound ofthis invention. Any effective nontoxic dose of 2-deoxyguanosine can beused, typically from about 0.5 to about 50 mg/kg per day will beadministered. By conjunctively applicants contemplate not only thosedosage forms which contain both 2-deoxyguanosine and a compound offormula 1, but also separate dosage forms. The compounds may also beadministered in separate dosage units.

The preferred route of administration is oral administration. For oraladministration the compounds can be formulated into solid or liquidpreparations such as capsules, pills, tablets, troches, lozenges, melts,powders, solutions, suspensions, or emulsions. The solid unit dosageforms can be a capsule which can be of the ordinary hard- orsoft-shelled gelatin type containing, for example, surfactants,lubricants, and inert fillers such as lactose, sucrose, calciumphosphate, and cornstarch. In another embodiment the compounds of thisinvention can be tableted with conventional tablet bases such aslactose, sucrose, and cornstarch in combination with binders such asacacia, cornstarch, or gelatin, disintegrating agents intended to assistthe break-up and dissolution of the tablet following administration suchas potato starch, alginic acid, corn starch, and guar gum, lubricantsintended to improve the flow of tablet granulations and to prevent theadhesion of tablet material to the surfaces of the tablet dies andpunches, for example, talc, stearic acid, or magnesium, calcium, or zincstearate, dyes, coloring agents, and flavoring agents intended toenhance the aesthetic qualities of the tablets and make them moreacceptable to the patient. Suitable excipients for use in oral liquiddosage forms include diluents such as water and alcohols, for example,ethanol, benzyl alcohol, and the polyethylene alcohols, either with orwithout the addition of a pharmaceutically acceptably surfactant,suspending agent, or emulsifying agent.

The compounds of this invention may also be administered parenterally,that is, subcutaneously, intravenously, intramuscularly, orinterperitoneally, as injectable dosages of the compound in aphysiologically acceptable diluent with a pharmaceutical carrier whichcan be a sterile liquid or mixture of liquids such as water, saline,aqueous dextrose and related sugar solutions, an alcohol such asethanol, isopropanol, or hexadecyl alcohol, glycols such as propyleneglycol or polyethylene glycol, glycerol ketals such as2,2-dimethyl-1,3-dioxolane-4-methanol, ethers such aspoly-(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant such as a soap or adetergent, suspending agent such as pectin, carbomers, methylcellulose,hydroxypropylmethylcellulose, or carboxymethylcellulose, or emulsifyingagent and other pharmaceutical adjuvants. Illustrative of oils which canbe used in the parenteral formulations of this invention are those ofpetroleum, animal, vegetable, or synthetic origin, for example, peanutoil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil,petrolatum, and mineral oil. Suitable fatty acids include oleic acid,stearic acid, and isostearic acid. Suitable fatty acid esters are, forexample, ethyl oleate and isopropyl myristate. Suitable soaps includefatty alkali metal, ammonium, and triethanolamine salts and suitabledetergents include cationic detergents, for example, dimethyl dialkylammonium halides, alkyl pyridinium halides, and alkylamines acetates;anionic detergents, for example, alkyl, aryl, and olefin sulfonates,alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates;nonionic detergents, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers; andamphoteric detergents, for example, alkyl-beta-aminopropionates, and2-alkylimidazoline quarternary ammonium salts, as well as mixtures. Theparenteral compositions of this invention will typically contain fromabout 0.5 to about 25% by weight of the active ingredient in solution.Preservatives and buffers may also be used advantageously. In order tominimize or eliminate irritation at the site of injection, suchcompositions may contain a non-ionic surfactant having ahydrophilelipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5 to about15% by weight. The surfactant can be a single component having the aboveHLB or can be a mixture of two or more components having the desiredHLB. Illustrative of surfactants used in parenteral formulations are theclass of polyethylene sorbitan fatty acid esters, for example, sorbitanmonooleate and the high molecular weight adducts of ethylene oxide witha hydrophobic base, formed by the condensation of propylene oxide withpropylene glycol.

Aerosol or spray compositions containing the compounds of this inventioncan be applied to the skin or mucous membranes. Such compositions maycontain a micronized solid or a solution of a compound of formula 1 andmay also contain solvents, buffers, surfactants, perfumes,anti-microbial agents, antioxidants, and propellants. Such compositionsmay be applied by means of a propellant under pressure or may be appliedby means of a compressible plastic spray bottle, a nebulizer, or anatomizer without the use of a gaseous propellant. A preferred aerosol orspray composition is a nasal spray.

The active ingredient may also be administered by means of a sustainedrelease system whereby the compound of formula 1 is gradually releasedat a controlled, uniform rate form an inert or bioerodible carrier bymeans of diffusion, osmosis, or disintegration of the carrier during thetreatment period. Controlled release drug delivery systems may be in theform of a patch or bandage applied to the skin or to the buccal,sublingual, or intranasal membranes, an ocular insert placed in the culde sac of the eye, or a gradually eroding tablet or capsule or agastrointestinal reservoir administered orally. Administration by meansof such sustained release delivery systems permits the tissues of thebody to be exposed constantly for a prolonged time period to atherapeutically or prophylactically effective dosage of a compound offormula 1. The unit dosage of the compound administered by means of asustained release system will approximate the amount of an effectivedaily dosage multiplied by the maximun number of days during which thecarrier is to remains on or in the body of the host. The sustainedrelease carrier may be in the form of a solid or porous matrix orreservoir and may be formed from one or more natural or syntheticpolymers, including modified or unmodified cellulose, starch, gelatin,collagen, rubber, polyolefins, polyamides, polyacrylates, polyalcohols,polyethers, polyesters, polyurethanes, polysulphones, polysiloxanes, andpolyimides as well as mixtures and copolymers of these polymers. Thecompounds of formula 1 may be incorporated in the sustained releasecarrier in a pure form or may be dissolved in any suitable liquid orsolid vehicle, including the polymer of which the sustained releasecarrier is formed.

Another aspect of this invention is the use of the purine nucleosidephosphorylase inhibitors of formula I in conjunctive therapy topotentiate the efficacy of antiviral nucleoside analogs which wouldotherwise become subject to the enzymatic action of purine nucleosidephosphorylase.

In particular, this invention embraces the use of a compound of FormulaI in conjunctive therapy for the treatment of retroviral infections,especially in humans, particularly human immunodeficiency virus.Particularly preferred 2', 3'-dideoxy purine nucleosides are2'3'-dideoxyadenosine, 2', 3'-dideoxyguanosine, 2',3'-dideoxythioinosine and 2', 3'-dideoxyinosine.

The potentiation of the anti-retroviral effect of a dideoxypurinenucleoside, e.g. of Formula I, by a PNP inhibitor can be determined,e.g. in cell cultures (e.g. H9 cells, ATH8 cells) exposed to aretrovirus (e.g. HIV) according to methodology well known in the art,such as described in Proc. Nat. Acad. Sci, U.S.A., 83, 1911 (1986). Thepotentiation can also be determined in vivo (e.g. in rats) by measuringthe increase in the plasma level of the dideoxypurine nucleoside whichis achieved by prior or simultaneous administration of the particularPNP inhibitor, according to methodology well known in the art.

The two active ingredients (2', 3'-dideoxypurine nucleoside and PNPinhibitors) may be administered simultaneously by the same or differentroutes, in the same or different formulations or may be administered atdiscrete points in time provided that there is effective PNP inhibitionwhen the 2', 3'-dideoxypurine nucleoside is present. The extent to whichthis time separation of administered active agents can be accomplisheddepends upon the amount of available PNP and the rate at which the PNPinhibitor is itself degraded. For these reasons, the preferred dosage isin divided doses two to four times a day, most preferably with bothagents being administered simultaneously.

It is known that purine nucleoside derivatives administered as antiviralagents are subject to a purine nucleoside phosphorylase catalysis whichundesirably alters the efficacy of such agents. Indeed, the catalysismay lead to untoward side effects. It is also known that antiviralcompounds which, per se, are not subject to purine nucleosidephosphorylase will, (either by mechanisms well understood e.g.,enzymatic action of adenosine deaminase or by mechanisms not understood)become subject to the action of purine nucleoside phosphorylase, andthus the antiviral effectiveness of this type of compound will similarlybe altered. Thus, for the purpose of this aspect of the invention bothtypes of antiviral agents are embraced by the term antiviral agentssubject to purine nucleoside phosphorylase.

This aspect of the invention may alternatively be expressed as: in theprocess for treating viral infections with antiviral agents subject tonucleoside phosphorylase, the improvement which comprises conjunctivelyadministering a therapeutically effective amount of a purine nucleosidephosphorylase inhibitor, particularly those inhibitors embraced hereinby those compounds embraced within the scope of the generic scope offormula I.

In this aspect of the invention, the term "antiviral" includes thetreatment of viruses and diseases caused thereby--commonly known to beamenable to treatment with nucleoside analogs such as, for example, theHIV viruses imputed to be causative factors in AIDS, hepatitis B virusand herpes.

Particular antiviral agents of current interest for which enhancedantiviral efficacy would be achieved with conjunctive therapy with thePNPase inhibitors are such compounds as

(a) dideoxy nucleosides of the formula ##STR31## wherein

    ______________________________________                                        R.sub.1, R.sub.2, X                                                                      Name             Target Virus                                      ______________________________________                                        OH, H, H   dideoxyinosine   HIV                                               OH, NH.sub.2, H                                                                          dideoxyguanosine HIV, Hepatitis B                                  OH, NH.sub.2, F                                                                          3'-F-dideoxyguanosine                                                                          HIV                                               OH, NH.sub.2, N.sub.3                                                                    3'-azidodideoxyguanosine                                                                       HIV, Hepatitis B                                  NH.sub.2, NH.sub.2, H                                                                    dideoxydiamino purine                                                                          HIV, HBV                                                     riboside                                                           NH.sub.2, H, H                                                                           dideoxyadenosine HIV                                               NH.sub.2, NH.sub.2, N.sub.3                                                              3'-azidodideoxydiamino-                                                                        HIV, HBV                                                     purine riboside                                                    ______________________________________                                    

The last three compounds are first substrates of adenosine deaminase,

(b) dideoxy dehydro nucleosides of the formula ##STR32## wherein

    ______________________________________                                        R.sub.1, R.sub.2                                                                        Name             Target Virus                                       ______________________________________                                        OH, H     dideoxydehydro inosine                                                                         HIV                                                OH, NH.sub.2                                                                            dideoxydehydro guanosine                                                                       HIV                                                NH.sub.2, H                                                                             dideoxydehydro adenine                                                                         HIV                                                ______________________________________                                    

(c) dioxolane purine derivatives of the formula ##STR33## wherein thevariations of R₁ and R₂ wherein R₁ is OH, or NH₂ and R₂ is H or NH₂,said compounds being targeted for HIV and HBV (i.e., hepatitis B), and

(d) oxetane type derivatives of the formula ##STR34## wherein R₁ is OHor NH₂ and R₂ is H or NH₂. The target viruses being HIV.

It is to be noted that the presently two best antiviral agents forhepatitis B (HBV) are dideoxyguanosine (ddGuo) and 2,6-diimino dideoxypurine riboside which is a "prodrug" of dideoxyguanosine.

Particularly useful compounds of this invention are the PNP inhibitorsof the formula ##STR35## wherein R₃ is H, Ar is 2,3-thiophene, 2,5-furanor 3,4-furan, and Z" is --CH₂ CF₂ -P(OH)₂, and wherein Ar is 1,2-phenyland

R₃ is H or NH₂ and Z" is:

CH₂ CF₂ PO(OH)₂,

CH₂ CHFPO(OH)₂,

CHFCF₂ PO(OH)₂,

CHOHCF₂ PO(OH)₂,

CH═CFPO(OH)₂,

CH═CHPO(OH)₂, or

COCF₂ PO(OH)₂.

As is true for most classes of chemo therapeutic agents, certainsub-generic and certain specific embodiments exhibit a more beneficialprofile than others. In this class of PNP inhibitors of formula I thosepreferred compounds are compounds wherein R₁ is OH, R₂ is NH₂, R₃ is Hor NH₂, Ar is 1,2-phenyl or 2,3-furan or thiophen, R₅ and R₆ are H.Preferred Z moieties are those of (a) wherein X and Y are F, R₄ is H andR'₄ is H or F, those of (b) wherein n is zero and X and Y are both H,those of (c) wherein X is F and Y is F or H, those of (d) wherein X is Fand Y is F or H, those of (e) wherein X and Y are F and R₇ and R₈ are H.The preferred specific compounds are those final products of examples 1to 10, as well as the 3-position amino analogs thereof.

What is claimed is:
 1. A method of suppressing the immune system in apatient in need thereof which comprises administering to the patient atherapeutically effective amount of a compound of the formula ##STR36##the tautomeric forms thereof, and the pharmaceutically acceptable saltsthereof, whereinR is ##STR37## with Ar being a R₉ -substituted phenyl,to which its adjacent CH₂ moiety is bonded to one ring carbon atom andthe Z moiety is bonded to a second ring carbon atom of the Ar moiety, Zis a moiety selected from the group consisting of sub-types (a), (b),(c), (d), or (e) wherein (a) is ##STR38## (b) is ##STR39## (c) is##STR40## (d) is --C.tbd.C--, and (e) is ##STR41## wherein n is aninteger of 1 to 5 or zero; R₁ is --OH or --SH;R₂ is H or --NH₂ ; R₃ isH, --NH₂, --OH, or --NH--NH₂ ; R₄ is H; R'₄ is H, OH or F, or R₄ andR'₄, together with the carbon atom to which they are attached, form aketo moiety; R₅ is C₁₋₆ alkyl or H; R₆ is C₁₋₆ alkyl or H; R₇ is H, F,or C₁₋₄ alkyl; R₈ is H, F, or C₁₋₄ alkyl; R₉ is H, Cl, Br, C₁₋₆ alkyl,C₁₋₆ alkoxy, OH, NH₂ or CH₃ ; X and Y are H, F or Cl, with the provisothat when n is zero, X and Y are both H.
 2. A method according to claim1 wherein said compound is selected from the group consistingof[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoroethyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1-fluoroethenyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1,2-trifluoroethyl]phosphonicacid,2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2-hydroxy-1,1-difluoroethyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2,2-dihydroxy-1,1-difluoroethyl]phosphonicacid,[3-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenoxy]-1,1-difluoropropyl]phosphonicacid,[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenoxy]methylphosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoro-2-propenyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]ethynyl]phosphonicacid, and[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]ethenyl]phosphonicacid.
 3. A method of inhibiting purine nucleoside phosphorylase in apatient in need thereof which comprises administering to the patient aneffective amount of a compound of the formula ##STR42## the tautomericforms thereof, and the pharmaceutically acceptable salts thereof,whereinR is ##STR43## with Ar being a R₉ -substituted phenyl, to whichits adjacent CH₂ moiety is bonded to one ring carbon atom and the Zmoiety is bonded to a second ring carbon atom of the Ar moiety, Z is amoiety selected from the group consisting of sub-types (a), (b), (c),(d), or (e) wherein (a) is ##STR44## (b) is ##STR45## (c) is ##STR46##(d) is --C.tbd.C--, and (e) is ##STR47## wherein n is an integer of 1 to5 or zero; R₁ is --OH or --SH;R₂ is H or --NH₂ ; R₃ is H, --NH₂, --OH,or --NH--NH₂ ; R₄ is H; R'₄ is H, OH or F, or R₄ and R'₄, together withthe carbon atom to which they are attached, form a keto moiety; R₅ isC₁₋₆ alkyl or H; R₆ is C₁₋₆ alkyl or H; R₇ is H, F, or C₁₋₄ alkyl; R₈ isH, F, or C₁₋₄ alkyl; R₉ is H, Cl, Br, C₁₋₆ alkyl, C₁₋₆ alkoxy, OH, NH₂or CH₃ ; X and Y are H, F or Cl, with the proviso that when n is zero, Xand Y are both H.
 4. A method according to claim 3 wherein said compoundis selected from the group consistingof[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoroethyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1-fluoroethenyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1,2-trifluoroethyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2-hydroxy-1,1-difluoroethyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-2,2-dihydroxy-1,1-difluoroethyl]phosphonicacid,[3-[2-[(2-amino-1,6-dihydro-6-chloro-9H-purin-9-yl)methyl]phenoxy]-1,1-difluoropropyl]phosphonicacid,[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenoxy]methylphosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]-1,1-difluoro-2-propenyl]phosphonicacid,[2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenyl]ethynyl]phosphonicacid, and [2-[2-[(2-amino-1,6-dihydro-6-oxo-9H-purin-9-yl)methyl]phenylethenyl]phosphonic acid.